Other Links

Campylobacter: Unmasking the
Secret Genes of a Food-Poisoning Culprit

Campylobacter bacteria are the
number-one cause of food-related
gastrointestinal illness in the
United States. To learn more
about this pathogen, ARS scientists
are sequencing multiple Campylobacter
genomes. This scanning electron
microscope image shows the
characteristic spiral, or corkscrew,
shape of C. jejuni cells
and related structures. (K11505-1)

The "juice" that always seems to leak out of those packages
of fresh chicken you bring home from the supermarket can make a big
mess on your kitchen counter. But more importantly, the juice can pose
a hazard to your health. Nasty microbes called Campylobacter jejuni
can live in that liquid and on the skin of fresh, uncooked poultry.

Thoroughly cooking chickenby grilling, frying, roasting, or bakingkills
this food-poisoning microbe. But if you accidentally splash some of
the raw juice on food that you'd planned to eat uncooked, such as leafy
greens for a fresh salad, you'd be wise to throw them out. Here's why:
If the microbe takes hold on those greens, as it is very adept at doing,
you might be in for a case of campylobacteriosis food poisoning that
you won't soon forget.

Campylobacter is thought to be the leading cause of bacterial
food poisoning in humans and is likely the perpetrator of more than
400 million cases of diarrhea every year. Though being careful when
you handle raw poultry should help keep you safe, ARS
researchers want to do more to zap this microbial menace before it reaches
your home.

Microarrays, or gene chips,
enable scientists to get a
quick look at thousands of
genes in a single experiment.
Here, technician Sharon Horn
monitors robotic equipment as
it imprints Campylobacter
microarrays on glass slides. (K11465-1)

At Albany, California, scientists in the ARS Produce Safety and Microbiology
Research Unit are making key advances in the international effort to
clobber Campylobacter. The California team, based at the Western
Regional Research Center, is focusing on Campylobacter's genes.

Why the interest in the microbe's genetic makeup? Because investigating
genes may lead to discovery of faster, more reliable ways to detect
the microbe in samples from humans and other animals, food, and water.

In addition, gene-based research opens the door to simpler, less-expensive
tactics for distinguishing look-alike species and strains of Campylobacter
and its close relatives, such as the Arcobacters. This will enable
experts to quickly finger culprit microbes in food poisoning outbreaks.

Finally, the studies may lead to innovative, environmentally friendly
techniques to circumvent the genes that make C. jejuni strains
so successful in causing human gastrointestinal upset and in some cases
paralysis or even death.

Technician Guilin Wang sets
the conditions for operation
of an automated robotic system
for purifying DNA. High-quality
DNA is required for spotting
onto glass slides for microarray
experiments. (K11476-1)

Working with the Institute for Genomic Research, Rockville, Maryland,
the Albany scientists have decoded the makeup, or sequence, of all the
genes and other genetic material in a specially selected strain of C.
jejuni.

This research represents the first time that a C. jejuni strain
from a farm animalin this case, a market chickenhas been
sequenced. That's important, notes research leader Robert E. Mandrell,
because chicken is the leading source of the bacterium in food. Earlier
C. jejuni genome sequencing, performed elsewhere, was based on
a specimen from a gastroenteritis patient and was lacking key features,
such as the ability to colonize chickens, Mandrell says.

The next step: Zero in on specific genes. "We're particularly
interested in the genes that make Campylobacter so viable and
virulent," says ARS molecular biologist William G. Miller. They're
targeting, for instance, genes that carry the code for making oligosaccharides.
These compounds likely enable the microbe to stick like glue to chicken
skin in the poultry processing plant even though the birds are bathed
and rinsed with chlorinated water. The oligosaccharides might be important
in invading and colonizing the human body, as well, Miller notes.

Technician Sharon Horn and
microbiologist William Miller
prepare samples of Campylobacter
for automated analysis of the
structure, or sequence, of the
DNA. The colored peaks on the
computer screen show the sequence
of a DNA sample from an
earlier run.(K11472-1)

With this genome sequence information in hand, the scientists are
developing microarrays, or gene chips, that make possible a quick look
at thousands of genes in a single experiment. For these analyses, robotic
equipment precisely places pieces of the pathogen's DNA in an array
of infinitesimally small droplets on glass microscope slides.

"We build and use these microarrays to compare and contrast DNA
of various Campylobacter samples," explains microbiologist
Craig T. Parker. "We're also using microarrays to get a snapshot
of genes in action so that we can see when genes are turned on or off."
For example, Parker is pinpointing the genes that are active in helping
Campylobacter overcome our bodies' protective actions. By tracking
the action of the microbes' genes, Parker and co-investigators may be
able to determine a way to derail them.

Research leader Robert Mandrell
(left) and microbiologist Craig
Parker, both of the Produce
Safety and Microbiology Research
Unit, examine an image of the
results of a microarray experiment
comparing over 1,700 genes ofCampylobacter jejuni strains
from farm animals and humans.(K11462-1)

Though C. jejuni has grabbed center stage because of its known
virulence, its relatives are also of interest. The Albany studies of
C. coli, C. lari, and C. upsaliensis, for example,
are attracting the attention of member nations in a three-continent
collaboration called "Campycheck," formed to evaluate the
importance of these lesser-known or newly emerging species. The Albany
scientists and colleagues from the ARS Richard B. Russell Agricultural
Research Center, Athens, Georgia, are advisors to Campycheck.

In clinical laboratories, these less-studied pathogens may inadvertently
be killed by the antibiotics used to identify the better-known ones.
The likely result? An inaccurate picture of their prevalence and virulence.
Campycheck may yield a detailed, accurate picture.

The Campylobacter studies in the United States and abroad might
never completely eliminate the need for careful handling of raw poultry
in our homes or the kitchens of school cafeterias, fine restaurants,
and other eateries. But the research can reduce our chances of ever
encountering this unruly microbe.By Marcia
Wood, Agricultural Research Service Information Staff.

This research is part of Food Safety, an ARS National Program (#108)
described on the World Wide Web at www.nps.ars.usda.gov.

Here's the puzzle: You have two samples of what seem to be the food-poisoning
microbe Campylobacter jejuni. A quick look at the specimens with
a microarray assay (see main story) shows no immediately apparent differences
in their genes. But when you expose pigletsanimals susceptible
to this microbeto the bacteria, one strain makes the animals ill,
while the other affects them only mildly.

Why the difference?

ARS food safety researchers Craig T. Parker, at Albany, California,
and colleague Michael E. Konkel at Washington State University in Pullman,
are designing a series of experiments that should enable them to find
out. What's more, their work may help other scientists who are investigating
the virulence of other major foodborne pathogens.

Even though their preliminary microarray scan failed to reveal significant
differences in the C. jejuni specimens' DNA, this technology
offers another optionone that allows them to delve more deeply.

Instead of beginning with the microbe's DNA, these followup assays
begin with RNAgenetic material that's formed when the DNA, or
genes, becomes active.

In these tests, the scientists will place the two strains in petri
dishes with colonies of a type of human intestinal cell. Called epithelial
cells, they're the target of real-life Campylobacter attacks.
The researchers will take samples of the two strains at successive intervals,
looking for changes in RNA that occur over time. RNA extracted from
the strains provides tell-tale evidence of genes that went into action.
The work is much like that of police detectives who analyze evidence
to reconstruct what really happened at a crime scene. The scientists
use an enzyme called reverse transcriptase to match up the RNA to a
version of the DNA from which it originated. Then, they use the microarray
assay to discern the differences between that DNA and the microbe's
DNA as it existed at the outset of the experiment. The comparison should
reveal genes that were activated in the attack and genes that remained
silent.

In earlier work at Pullman, collaborator Konkel uncovered one such
C. jejuni gene. Named ciaB, short for Campylobacter
invasion antigen B, it cues the microbe to secrete a similarly named
protein, CiaB, which apparently plays a crucial role in enabling the
bacterium to penetrate epithelial cells. Though undoubtedly key to C.
jejuni's invasions, it is unlikely to act alone. The West Coast
scientists expect to uncover other genes that will lead them into the
dark heart of Campylobacter's virulence.By Marcia
Wood, Agricultural Research Service Information Staff.

"Campylobacter: Unmasking the Secret Genes of a Food-Poisoning
Culprit" was published in the October
2004 issue of Agricultural Research magazine.